1. Field of the Invention
The present invention relates to an asymmetric liposome for highly efficient encapsulation of nucleic acid and hydrophilic anionic compound and a preparation method thereof.
2. Description of the Related Art
Drug delivery system (DDS) that effectively delivers necessary amounts of drug without causing side effect and provides maximized efficacy and effect of the drug is one of the high value core technologies with high possibility of success which can create economic profit comparable to that made by new drug development and improve quality of patients' lives.
Solubilization of non-soluble drug, which facilitates drug absorption, is one of the core technologies of DDS and is currently considered the most reasonable way to decrease cost for developing new substances and increase value of pharmaceutical products. Particularly in South Korea, environments are not supportive to developers of new drugs. The advanced solubilization technology will create tremendous values with relatively small investment by developing modified new drugs (KAIST Annual Report on Technology Trend, 2004).
Liposome is one of the most widely used substance that is used in the delivery of genes or drugs.
Liposome is a lipid-bilayer vesicle formed of phospholipid of amphipathic lipid cell layer as a main component, and used for drug delivery by encapsulating soluble drug in aqueous compartment or carry the hydrophobic drug in lipid-bilayer. The layer structure of the liposome is similar to that of cell layer and has small toxicity. Accordingly, drug delivery is possible via fusion with cells or introduction into cell. Further, depending on combination, properties including sizes, surface potential or chemical modifications are adjustable. Further, the excellent biocompatibility is the reason for active efforts to develop liposome as a drug delivery substance (Bangham, Torchilin, V. P. 2005, Nat. Rev. Drug Discov., 4; 145).
An example of commercialized liposomal drug is Ambisome® developed to treat indication of mycotic infection by the association of Gilead (USA) and Fujisawa (Japan) (FIG. 1). Ambisome® is prepared by introducing amphotericin, which is the mycotic infection treating agent, into the unilamellar bilayer of approximately 100 nm liposome by the hydrophilic attraction with long-chain alkyls of phospholipid, and adding components including hydrogenated soy phosphatidylcholine (HSPC), cholesterol, distearoylphosphatidyl glycerol (DSPG), antioxidant, anti-coagulating agent, or the like and then lyophillizing (US FDA product information Amphotericin).
Like other particulate DDS for use in systemic circulation, liposomes have shortcomings that these are lost in the circulatory system due to phagocytosis of macrophagocyte prevalently existing in the livers or spleens due to adsorption of proteins in the blood or spillage of the drug from the liposomes during circulation in the blood. The phagocytosis of macropagocyte is of particular concern, because this induces adsorption of opsonic protein on the surfaces of liposomes. To solve the above-mentioned shortcomings, it was suggested to suppress adsorption of opsonic protein by using phospholipid-PEG derivative having PEG(poly[ethylene glycol]) bound to the terminus of phosphatidylethanolamine, or coating the surface of the prepared liposome with PEG or multisaccharides. These liposomes prolong drug circulation time in the blood by reducing loss of drug by the phagocytosis of macropagocyte, and furthermore, enhances arrival rate of the drug to the targeting organs. The 100 nm sized PEG-liposome technology developed by Alza (USA), so-called stealth liposome, was commercialized in the form of liposomal anticancer agent with increased circulation time in the blood. Ortho Biotech (USA) commercialized Doxil, the result of applying the stealth-liposome technology to doxorubicin, the anthracyclinc family anticancer agent (US FDA product information Doxil).
The nucleic acid such as antisense RNA or siRNA has gained attention as an important tool for the treatment of cancer, genetic disease, infectious disease, autoimmune disorder, etc., for its property to inhibit expression of specific proteins in living body (Novina and Sharp, Nature, 430, 161-164, 2004). However, many researches are conducted to overcome the shortcomings of using nucleic acid such as siRNA, such as difficulty of direct delivery into cells and easy disruption by enzyme in the blood.
Recently, the method for delivering nucleic acid into cells include mixing with positively-charged lipid (lipid-DNA complex or lipoplex) and mixing with polymer (polymer-DNA complex or polyplex) (Hirko et al., Curr. Med. Chem., 10, 1185-1193, 2003; Merdan et al., Adv. Drug. Deliv. Rev., 54, 715-758, 2002; Spagnou et al., Biochemistry, 43, 13348-13356, 2004). Lipid-DNA complex is widely used at a cellular level because this binds to nucleic acid to facilitate delivery of nucleic acid into the cell. However, lipid-DNA complex in many cases induces inflammation in vivo when injected locally (Filonand and Phillips, Biochim. Biophys. Acta, 1329, 345-356, 1997), and accumulates in the organs on the passage when intravascular-injected (Ren et al., Gene Therapy, 7, 764-768, 2000). On the contrary, because phospholipid existing in vivo is neutral or negatively-charged, it has considerably low affinity to nucleic acid.
One suggestion to solve the above-mentioned drawback is to join another lipid such as cholesterol to the sense strand of siRNA for delivery thereof. However, because these complexes are joined with lipoprotein such as LDL and HDL in the blood and delivered, the complexes mainly have liver tissue specificity (Soutschek et al., Nature, 432, 173-178, 2004; Wolfrum et al., Nat. Biotechnol., 25, 1149-1157, 2007).
Further, polyplex, in which nucleic acid is joined with cationic polymer, has the shortcoming of low delivery rate to an intended tissue due to reasons such as removal by the complement, etc. (Plank et al., Human Gene Ther., 7, 1437-1446, 1996; Chui et al., Chem. Biol., 11, 1165-1175, 2004; Elmen et al., nucleic Acids Res., 33, 439-447, 2005).
Additionally, siRNA exposure acts as an agonist to the toll-like receptor and thus non-specifically increases interferon alpha expression (Hornung et al., Nat. Med., 11, 263-270, 2005) or induces non-specific innate immune reaction (Judge et al., Nat. Biotech., 23, 457-462, 2005).
It was suggested that siRNA stability can be improved and non-specific immune reaction can be removed by substituting 2′-OH of siRNA with 2′-F, 2′-OMe and 2′-H (Morrissey et al., Hepatology, 41, 1349-1356, 2005), but the cost for synthesis is huge and it is necessary to prepare lipoplex and polyplex to ensure delivery.
Because hydrophilic anionic drug hardly penetrates cells, a medicine developed with this hydrophilic anionic drug is generally made in the form of prodrug capable of cell permeation. Further, hydrophilic anionic fluorescence substance such as calcein, indocyanine green, or chlorotoxin (Cy5.5) can be used as an important imaging tool to measure movement of liposome in the body.
To encapsulate nucleic acid with strong anionic property into liposome, the lipid mixture containing cationic lipid is generally utilized. Among the currently developed methods, the methods with relatively high encapsulation rate of polymer nucleic acid include agitation using ultrasonic waves with the use of EPC/CH/lysine-DPPE (Encapsulation rates of various nucleic acids, 60-100%) (Puyal et al., Eur. J. Biochem. 15, 697-703, 1995), detergent dialysis using DOPE/PEG-Cer/DODAC (4-10 Kbp, encapsulation rate, 60-70%)(Wheeler et al., Gene Ther., 6, 271-281, 1999), ethanol dialysis using (DODAP/Chol/DSPC/PEG-cer or DSPC/Chol/PEG-C-DMA/DLinDMA (ODN encapsulation rate 50% or siRNA encapsulation rate 93%) (Semple, 2001 Biocheim. Biophys. Acta, 1510; 152-166; Morrissey, 2005, Nat. Biotech., 23; 1002-1007), hydrolysis of lyophilized matrix using DOTAP/Chol/DOPE/PEG-cer (siRNA encapsulation rate, 95%) (Wu et al., Pharm. Res., 26, 512-522, 2009) and many others. However, because all these methods encapsulate nucleic acid by use of one common composition of lipoplex in the preparation thereof, it is difficult to encapsulate low molecular hydrophilic anionic substance effectively.
Accordingly, the inventors of the present invention prepared asymmetric liposome having an interior consisting of a cationic lipid which is toxic internally but is advantageous for the purpose of encapsulating nucleic acid and hydrophilic anionic compound, and has a small head group to enable desirable placement within liposome, and an exterior consisting of a neutral and anionic lipid with similarity to cell surface and thus is less toxic, and completed the present invention by confirming that the liposome prepared as explained above can encapsulate siRNA and hydrophilic anionic compound.